Electro-blotting of electrophoretically resolved fluroescent-labeled saccharides and detection of active structures with protein probes

ABSTRACT

Saccharide in mixtures are separated into groups or individual saccharides and tested for their affinity for particular proteins. The method of the invention is carried out by conjugating saccharides in a mixture with 4-amino-1-naphthalene sulfonic acid (ANSA) to form conjugates. The saccharide/ANSA conjugates are subjected to electrophoretic resolution within gels. The resolved bands of material are electro-blotted onto charged nylon membranes to provide a stable record of the electrophoretic separation. The blots on the nylon membranes are brought into contact with particular proteins in order to determine the binding affinity of these particular proteins to particular saccharides on the nylon membrane. The proteins are preferably bound to labels so that the binding of the proteins to the saccharides can be easily detected.

CROSS-REFERENCE

This application is related in part to two other co-pending U.S.applications filed concurrently with the present application on Feb. 16,1990. One related application is Ser. No. 07/481,361, which applicationis entitled "Two-Dimensional Electrophoretic Separation ofCarbohydrates" invented by Brian K. Brandley, a co-inventor of thepresent invention; the other application is Ser. No. 07/483,043, whichis entitled "Fluorescent Tag for Sugar Electrophoresis" also invented byBrian K. Brandley and Michael Tiemeyer, who are co-inventors working inthe same research organization as the present inventors with anobligation to assign the invention to the same entity. Theabove-referenced applications are each incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

This invention relates generally to separation techniques such aselectrophoresis and electro-blotting which allow for the separation ofparticular materials and then testing of those materials for activityutilizing labeled probes. More particularly, the invention relates toutilizing electrophoresis followed by electro-blotting in order toseparate mixtures of saccharides containing very small amounts offluorescent labeled saccharides and then determining the activity ofsuch saccharides by using labeled protein probes.

BACKGROUND OF THE INVENTION

Electrophoresis is a well known technique for the separation of acharged species by utilizing their differences in rate of migrationunder the influence of an electrical field. The procedure has provedinvaluable for the resolution and isolation of complex biologicalsubstances such as enzymes, serums, carbohydrates, proteins, DNA andRNA. Most analytical electrophoresis methods are based onzone-electrophoresis in which a thin zone of a sample material isapplied to the electrophoretic medium. The electrophoretic migration ofthe sample components results in the formation of fractional zones.These zones can be examined and studied by applications of standardelectrophoretic practice such as fixing, staining and washing to removebuffers. Desirably, the electrophoretic media is a thin gel film coatedon a suitable support, commonly glass or plastic. Such an arrangementpermits the electrophoretic separation to be achieved in a minimum oftime with a maximum degree of resolution.

Various hydrophilic colloids, for example, starch, agarose and cellulosederivatives have been used in forming electrophoretic gel films, butpolyacrylamide is preferred. One reason for preferring polyacrylamide isthat gels can be prepared from it having a wide range of pore size. Thisis accomplished primarily by varying the ratio of acrylamide polymer tothe N, N', methylenebisacrylamide cross-linking reagent.

The resulting polyacrylamide gels provide high resolutionelectrophoretic separation of important biopolymers, for example,proteins and nucleic acids. In addition, the absence of ionized groupsin polyacrylamide gels render such gels suitable as an anticonvectionmedium for isoelectric focusing.

Once the electrophoretic techniques have been applied in order toseparate the materials in the gel, it is necessary to transfer theseparated materials from the gel to a support where they can be tested.A number of procedures are available for transferring theelectrophoretically resolved materials from the gel. One such procedureinvolves electro-blotting. This type of transfer procedure involvestransferring the resolved bands within the gel to a support matrix suchas a nitrocellulose sheet. The transfer is carried out by theapplication of an electric field and therefore is distinguishable from amore conventional alternative which involves the capillary transfer ofsuch materials usually used in techniques such as southern and northernblotting.

SUMMARY OF THE INVENTION

In accordance with the present invention mixtures of saccharides areresolved into groups and tested for their affinity to particularproteins. The process is carried out by conjugating the saccharides inthe mixture to a charged molecule and then subjecting the chargedconjugates to electrophoretic techniques in order to separate thedifferent saccharides or groups of saccharides from each other. Aftercarrying out electrophoretic resolution, the resolved bands within thegel are electro-blotted onto a membrane which is preferably a chargednylon membrane. The nylon membrane provides a stable record of theelectrophoretic separation in the gel. The membrane having the bands ofsaccharides thereon is then subjected to testing techniques with variouslabeled protein compounds. If the protein binds to the saccharidematerial, the binding can be detected by the probe and the saccharide isdetermined as being one of particular interest due to its bindingaffinity to the protein and can be further studied.

A primary object of the present invention is to provide a method forseparating mixtures of saccharides into closely related groups ofsaccharides and determining which groups of specific saccharides haveparticular binding affinities to particular proteins.

An advantage of the present invention is that large numbers of differentsaccharides each present in small amounts in mixtures can be readilyseparated and readily tested for their affinity to large numbers ofproteins.

A feature of the present invention is that it involves a uniquecombination of separation and probing techniques which can be readilyand efficiently carried out to obtain large amounts of information withrespect to particular saccharides.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis and usage as more fullyset forth below, reference being made to the accompanying generalstructural formulae forming a part hereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present method for separating and testing saccharides isdescribed, it is to be understood that this invention is not limited tothe particular saccharides, proteins or process steps described as suchcompounds and steps may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intending to be limiting sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used in the specification and the appendedclaims, the singular forms "a", "an" and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "a protein" includes mixtures of proteins, reference to "anoligosaccharide" or a saccharide includes reference to mixtures ofoligosaccharides or saccharides, and reference to "the electrophoreticprocessing step" includes a variety of similar steps of the typedescribed herein.

The first step of the present invention involves binding the saccharidemolecules to be tested to a charged molecule. This is done in order toallow for the mixtures of saccharides to be separated from each other byelectrophoretic techniques which require that the materials beingseparated have electrical charges thereon. In connection with thepresent invention, the saccharides are conjugated with4-amino-1-naphthalene sulfonic acid (hereinafter ANSA).

The 1-amino-4-naphthalene sulfonic acid (ANSA) used in connection withthe present invention has the following structure: ##STR1## The -NH₂amino group is indicated as being at the "1" position and the -SO₃ ⁼group is at the "4" position. The shared double bonds in each ringstructures provides the fluorescent character to the ANSA when the ANSAis exposed to U.V. light.

Depending on the type of saccharide in the mixture being resolved, theremay be additional -SO₃ ⁼ groups attached to the basic ring structure.The additional -SO₃ ⁼ groups provide additional charge to neutralsaccharides. If two -SO₃ ⁼ groups are present, the molecule is referredto as ANDA and if three are present ANTS, for example,(8-aminonaphthalene-1,3,6 trisulfonic acid).

After the saccharide/ANSA conjugates are formed, they are subjected tostandard electrophoretic techniques in order to resolve the differentsaccharides from each other. The electrophoretic resolution provides agel wherein the saccharides are separated from each other in differentbands along the length of the gel.

The separation techniques utilized in connection with the presentinvention have been found to work particularly well in connection withsmaller saccharides. More specifically, the gel electrophoresis has beenfound to be particularly useful in separating mixtures ofmonosaccharides, disaccharides, and trisaccharides. Conventionalprocedures are generally not capable of providing sufficient resolutionto separate away smaller saccharides into distinct bands. The additionof the ANSA group provides a sufficient amount of charge to allow forthe separation of the smaller saccharides into distinct groups but doesnot apply too much charge so that the charge quality overwhelms anyother quality of the saccharides and thus does not provide forresolution among different types of closely related saccharides.Further, the shared double bonds within the ring structures of the ANSAprovide for the fluorescent capability of the conjugates formed.Accordingly, when different bands of saccharides are separated away fromeach other, it is possible to visually view these bands simply by theapplication of ultraviolet light.

It is pointed out that some mono- and disaccharides can only be resolvedinto distinct groups via electrophoresis by using ANSA. Themonosaccharides include glucuronic acid, iduronic acid and galacfuronicacids. The disaccharides include:

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronicacid)-D-galactose;

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronicacid)-4-0-sulfo-D-galactose; and

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronicacid)-6-0-sulfo-D-galactose

The use of such a fluorescent tag provides a number of advantages overand above the use of other types of tags. For example, a fluorescent tagis substantially safer and less expensive than the use of a radiolabel.Further, the use of a fluorescent tag is substantially less cumbersomeand more efficient than the use of antibody-linked enzyme tags. Theseadvantages are obtained concurrently with the overall advantage ofproviding a tag which allows for greatly improved resolution especiallyas used in connection with smaller saccharide compounds.

The separated bands of conjugates within the gel are then transferred tothe surface of a membrane. A number of different types of membranesurfaces can be utilized in connection with the invention. However,nylon is preferable. The transfer of the saccharide/ANSA conjugates fromthe gel to the surface of the substrate is carried out by utilizingelectro-blotting techniques. The electro-blotting is carried out for asufficient period of time to allow substantial amounts of the conjugateswithin the gel to transfer to and bind to the surface of the substratethus providing a permanent record of the separated bands of conjugateson the surface of the membrane.

The electro-blotting procedures which can be used in connection with thepresent invention are procedures which are generally known to thoseskilled in the art. In general, a gel having the separated conjugatesthereon is placed in contact with a membrane surface. The membranesurface which is preferably a charged nylon surface is preferably firstwetted with a buffer in which the electro-blotting procedure will becarried out. The membrane surface may be coated with polyisobutylenemethylmethacrylate. What is arbitrarily chosen as the cathode side ofthe gel (i.e., ultimately towards the negative electrode when positionedin the electro-blotting tank) is placed in contact with the surface ofthe nylon substrate after the substrate has been moistened with theelectroblotting buffer. Any air bubbles between the gel and the nylonmembrane should be removed by gently pushing the nylon substrate againstthe gel using powder-free gloved fingers. A piece of nitrocellulose canbe placed on the opposite side of the gel and all of the air bubblesshould construct is then placed in the electro-blotting tank whichcontains a buffer solution and has an anode and a cathode therein. Thepower supply is then turned on and the power supply will draw theelectrically charged saccharide/ANSA conjugates out of the gel and ontothe charged surface of the nylon substrate. The transfer time isdependent somewhat on the thickness of the gel and the size of theconjugates being transferred to the nylon substrate. The transfer can bemonitored by viewing the transfer under U.V. light to insure completetransfer of all of the materials to the nylon substrate surface.Overnight transfer is reliable and convenient.

One of the surprising discoveries of the present invention is that thespecific bands of conjugates in the gel are even more clearly resolvedand distinguishable from each other when the transfer is made to thenylon substrate surface. While not wishing to be bound to anyparticularly theory, it is believed that greater resolution is obtainedon the nylon surface because of the diffusion of light in the gel whenthe conjugates are exposed to U.V. light. Regardless of the reason, ithas been found that distinct, separate bands of conjugates are formed onthe nylon substrate surface.

After the conjugates on the membrane surface have been secured to thesurface, the specific saccharides within each of the visually detectablegroups can then be tested for their affinity to other molecules. Thistesting is generally done by first forming conjugates of molecules to betested by binding such molecules to a label. For example, proteinmolecules are bound to a radiolabel. The conjugates of radiolabeledproteins are then brought into contact with the saccharide/ANSAconjugates on the surface of the membrane. If the proteins have anaffinity to the saccharides on the membrane, they will bind to thesaccharides, thus forming double conjugates, i.e., the saccharide/ANSAconjugates bind to the protein/label conjugates.

After the protein/label conjugates have been allowed to remain incontact with the membrane surface for a sufficient period of time toallow for complete binding, the membranes are washed thoroughly in orderto remove any unbound protein. After the unbound proteins are removed,the bound proteins, if any, are detected by utilizing the label attachedto the proteins by procedures such as radiography.

The separation methodology of the present invention can be utilized inorder to test a variety of different types of compounds for theiraffinity to the saccharides on the nylon substrate. For example, theinvention can be utilized in order to test the affinity of certainlectins for their affinity to the saccharides. Particular types ofantireceptor proteins known to be positioned on viruses and to beattachable to certain saccharides on cell surfaces can be tested.Further, the affinity of certain growth factor proteins can be tested.It is believed that the attachment of certain saccharides to growthfactor proteins can effect the activity of the growth factor protein invivo.

The molecules to be tested, such as the protein molecules to be testedfor their affinity to saccharides, must, of course, be bound to a labelwhich is later detectable. A variety of different types of labels knownto those skilled in the art can, of course, be used. For example, it ispossible to utilize radiolabels which are later detected by the use ofautoradiography. It is also possible to attach the protein molecules toan antibody which itself is bound to an enzyme such as horseradishperoxidase which can be detected by the addition of reagents which causea color change. Procedures for attaching the labels to the proteins orother molecules to be assayed are well known to those skilled in theart.

The following examples are provided so as to give those of ordinaryskill in the art a complete disclosure and description of how to carryout the processes of the invention and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in degrees centigrade, and pressure is at or nearatmospheric.

EXAMPLE 1

Charged saccharides in the form of heparin fragments from partialnitrous acid digests are reacted with ANSA to form conjugates. Thereaction is carried out in 10 to 100 mM sodium acetate buffer (pH 5.0).The heparin fragments are present in an amount of 0.01-1 umol/ml and arereacted with ANSA and sodium cyanoborohydride in a ten fold molar excesswith respect to the reducing end sugar of the heparin fragment. Thederivatized heparin fragments are then subjected to electrophoreticresolution in 40% acrylamide/5% bis gels, with a Tris/ glycine buffersystem (25 mM Tris, 195 mM glycine, pH 8.3). The electrophoretic gel isto be run at 300 volts for approximately 90 minutes. The gels are to beimmediately electro-blotted using a Biorad apparatus onto a Zetaprobemembrane (of the type commercially sold by Biorad). The electro-blottingis carried out utilizing standard techniques and 100 volts for one hourwith the same Tris/glycine buffer system. After completing theelectro-blotting, the Zetaprobe membranes are removed and air dried.

The drying membranes have the separated heparin fragments bound thereon.These membranes with the heparin bound thereon are stable on PBS atleast overnight. Accordingly, such blots can be blocked with 2% PVP40(Sigma) in PBS for 1 hour at room temperature and then probed withproteins (radioiodinated bFGF) overnight at 4° C., in PBS plus 2% PVP40.After allowing any binding to take place, the membrane are washed withthe same buffer three times, and then dried. Bound protein can bedetected by autoradiography. The ANSA provides a fluorescent label onthe heparin fragment will allow for the direct visualization of the blotand comparison of it with the autorad, i.e., the membrane having theradiolabeled proteins bound thereto.

EXAMPLE 2

Neutral saccharides in the form of tetrose derived from asialo-GM1 werecharged by reacting them with ANSA to form conjugates. The reactionconditions were 10kb/100 mM sodium acetate buffer (pH 5.0), asialo-GM1at 0.01-1 uM/ml, ANSA and sodium cyanoborohydride in tenfold molarexcess to the reducing end of the sugar. Derivatized saccharides wereresolved in 40% acrylamide/5% bis gels with a Tris/glycine buffer system(25 mM Tris, 195 mM glycine pH 8.3) run at 300 volts for approximately90 minutes. The gels were immediately electro-blotted using a Bioradapparatus onto Zetaprobe membranes (manufactured by Biorad) by standardtechniques (100 volts, 1 hour) with the same Tris/glycine buffer system.

It was found that the ANSA tag did not apparently impart sufficientcharge to keep these neutral saccharides stably associated with themembrane in buffer solutions. Accordingly, the membranes were firstwetted with hexanes, then soaked in 0.1 percent polyisobutylenemethylmethacrylate in hexanes for 45-60 seconds and were dried. Thesemembranes were probed in 50 mM buffers.

The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom which are inthe scope of the invention and that obvious modifications will occur toone skilled in the art upon reading this disclosure.

What is claimed is:
 1. A method of separating and analyzing saccharideswithin a mixture of saccharides, comprising the steps of:reacting thesaccharides in the mixture with 4-amino-1-naphthalene sulfonic acid inorder to form conjugates; subjecting the conjugates to electrophoreticseparation in a gel; electro-blotting the separated saccharides in thegel to the surface of a support membrane; and contacting labeled probeswith the membrane and determining binding affinity of the probes tosaccharides on the membrane.
 2. The method as claimed in claim 1,wherein the membrane is a nylon membrane.
 3. The method as claimed inclaim 1, wherein the mixture of saccharides is a mixture of heparinfragments obtained from partial nitrous acid digests.
 4. The method asclaimed in claim 1, wherein the saccharide mixture includes tetrosederived from asialo-GM1.
 5. The method as claimed in claim 1, whereinthe mixture of saccharides includes neutral oligosaccharides.
 6. Themethod as claimed in claim 5, further comprising:coating the surface ofthe membrane having the saccharides electro-blotted thereon withpolyisobutylene methylmethacrylate.
 7. The method as claimed in claim 1,wherein the mixture of saccharides includes saccharides selected fromthe group consisting of mono-saccharides, di-saccharides trisaccharides.8. The method as claimed in claim 1, wherein the labeled probe is alabeled protein probe.
 9. A method of resolving a mixture of saccharidesinto distinct bands of saccharides which are closely related oridentical, comprising the steps of:reacting the mixture of saccharideswith 1-amino-4-naphthalene sulfonic acid to form conjugates; subjectingthe conjugates to gel electrophoresis separation in an electrophoresisgel by the application of an electrical field for a sufficient period oftime to form separate bands in the electrophoresis gel; electro-blottingthe separate bands of saccharides in the electrophoresis gel onto amembrane surface; and exposing the membrane surface to ultraviolet lightto determine the bands of saccharides.
 10. The method as claimed inclaim 9, wherein the electro-blotting of the bands in theelectrophoresis gel is onto the surface of a nylon membrane.
 11. Themethod as claimed in claim 10, wherein the saccharide mixture comprisessaccharides selected from the group consisting of monosaccharides,disaccharides and trisaccharides.
 12. The method as claimed in claim 10,further comprising:contacting the membrane surface with labeled probesto determine the affinity of the probes to bind to saccharides on themembrane surface; and washing away any labeled probes not bound tosaccharides on the surface and detecting bound probes by their label.13. The method as claimed in claim 9, wherein the mixture of saccharidesincludes specific saccharides in an amount in the range of 1 to 10picomoles and wherein such saccharides present in an amount of 1 to 10picomoles are observable in distinct bands on the membrane surface whenviewed under ultraviolet light.
 14. The method as claimed in claim 13,wherein distinct bands of saccharides on the membrane surface includessaccharides in an amount of from 1 to 5 picomoles.
 15. A method ofseparating and analyzing saccharides within a mixture of saccharides,comprising the steps of:reacting the saccharides in the mixture with anamino-naphthalene sulfonic acid compound selected from the groupconsisting of 4-amino-1-naphthalene sulfonic acid and4-amino-1-naphthalene sulfonic acid compounds having an additionalsulfonic acid group attached thereto at the 5, 6, 7 or 8 positions, thereacting being carried out in order to form conjugates; subjecting theconjugates to electrophoretic separation in a gel; electro-blotting theseparated saccharides in the gel onto the surface of a support membrane;and contacting labeled probes with the membrane and determining thebinding affinity of the probes to saccharides on the membrane.